The studies described in this proposal address the cellular mechanisms responsible for secretion by cholangiocytes, the epithelial cells that line the lumen of intrahepatic bile ducts and account for ~40% of bile volume in man. Previous studies indicate that the secretory capacity of cholangiocytes undergoes dynamic changes in response to varying physiologic demands; and have identified multiple pools of intracellular vesicles that can be mobilized rapidly and are sufficient in size to replace up to 40% of the entire cholangiocyte plasma membrane within minutes. Further, the best evidence supports a model wherein one of these vesicular pools is enriched in ATP, and exocytosis leads to a local increase in extracellular nucleotides and initiation of a purinergic signaling cascade. These different vesicular pools are functionally linked to cellular ATP release, activation of purinergic signaling and selective opening of separateC/" channels in response to cAMP, cytosolic Ca2+, and cell volume. Accordingly, the Specific Aims are designed to further address the working hypothesis, which is unchanged, that trafficking (endocytosis and exocytosis) of membrane vesicles containing ion channels provides an early and essential mechanism for modifying the composition and conductance of the cholangiocyte plasma membrane and is essential for the secretory responses to cAMP, Ca2+, and cell volume. The Specific Aims continue to focus on 1) characterization of the cellular signals responsible for regulation of endocytosis and exocytosis; 2) assessment of the functional roles of small conductance K+ (SK2) channels in regulation of cholangiocyte volume and ductular secretion; and 3) definition of the cellular strategies for maintaining the multiprotein signaling complex responsible for apical ATP release, P2 receptor distribution and K+ and CI"secretion. To that end, we have developed combined imaging and biophysical approaches to measurement membrane dynamics and ion transport in real time; and to detection of cellular ATP release in both macroscopic and individual cell models. The long term goal is to define the cellular mechanisms involved in ductular secretion, and to identify the physiologic factors which contribute to bile formation through effects on duct cells. Thus, the findings continue to be directly relevant to diagnosis and management of a broad range of cholestatic disorders characterized by impaired cholangiocyte function; and to development of pharmacologic approaches to modify the volume and composition of bile through effects on duct cells